A study carried out at Johns Hopkins University has shown that pulverized bone can be combined with polycaprolactone (PCL) to create viable 3D printed cell scaffolds for bone replacment procedures. The study found that mixtures with more bone powder encouraged bone formation better, but that those with more PCL were stronger.

Each year, around 200,000 people require replacement bones in the head or face because of birth defects, trauma, or surgery. The conventional medical procedure for implanting these replacement bones is to remove part of the patient’s fibula, before chiseling it to the size and shape required. This, however, is far from a perfect solution: the procedure can cause leg trauma, and the sculpted piece from the relatively straight fibula rarely fits perfectly into the natural curvature of the face.

Luckily, scientists are slowly but surely developing a new way of creating replacement bone—potentially better bone, for that matter, which can fit perfectly into the target area. The new method involves 3D printing partially plastic scaffolds on which living cells can be placed. By 3D printing the replacement bone section, medical professionals can create a perfectly fitting implant, without necessarily taking bone from the fibula. Things aren’t completely straightforward though: cells on plastic scaffolds need some direction in order to start forming bone tissue. Another piece of bone would do the trick, but natural bones cannot easily be shaped. So what’s the solution?

A team of researchers at Johns Hopkins University has been performing experiments on bone-plastic composites, combining 3D printability with the organic framework required to promote natural bone growth. The main synthetic material used by the researchers in their concoction is polycaprolactone, an FDA-approved, biodegradable polyester with a low melting point (80 to 100 degrees Celsius or 176 to 212 Fahrenheit). PCL is strong, but does not encourage bone growth particularly well. Therefore, to turn PCL into a viable bone replacement material, the researchers added pulverized bone from the inside of cow knees.

“Bone powder contains structural proteins native to the body plus pro-bone growth factors that help immature stem cells mature into bone cells,” explained Warren Grayson PhD, associate professor of biomedical engineering at the Johns Hopkins University School of Medicine and senior author on the study. “It also adds roughness to the PCL, which helps the cells grip and reinforces the message of the growth factors.”

To assess the potential of the combined materials, the researchers tried mixing the PCL and bone powder in different ratios: five, 30, 70, and 85 percent bone powder blends were each developed. The most bone-heavy of those mixtures, the 85 percent, was not a success. The small amount of PCL meant that the material simply didn’t work as a 3D printable substance, with the required lattice shapes simply falling apart. “It was like a chocolate chip cookie with too many chocolate chips,” said Ethan Nyberg, a graduate student working on the project.

All of the other mixtures could be 3D printed with relative ease, so the next step involved testing the materials’ potential to induce bone growth. To do so, the researchers introduced human fat-derived stem cells, taken during a liposuction procedure, placed within a “nutritional broth”. After three weeks, cells grown on the 70 percent bone powder scaffolds were doing incredibly well, showing high rates of gene activity in three bone formation-indicative genes. The gene activity on these genes was hundreds of times higher when compared with the purely PCL scaffolds. The 30 percent bone powder scaffolds also showed good results, though less drastic than the 70 percent.

The next step was to introduce a key ingredient into the cell broth: beta-glycerophosphate, which would encourage the enzymes in the cells to deposit calcium, the primary mineral in bone. Again, the researchers were pleased with what they saw: the 70 percent bone scaffolds produced double the amount of calcium per cell than the purely PCL scaffolds, while the 30 percent scaffolds produced about 30 percent more than the PCL.

Clearly then, the 70 percent bone mixture had the edge over the 30 percent, and showed remarkably better results than a scaffold made of just PCL. But what difference would these results make in a real test case? The final stage of the experiment involved implanting these 3D printed bone-PCL composites into mice skulls, each of which contained large holes made experimentally. These holes were too large to heal without some kind of stimulation, but implanting the bone mixture caused new bone growth in each mouse over a 12-week period. CT scans showed that at least 50 percent more bone grew in scaffolds containing 30 or 70 percent bone powder, compared to the pure PCL.

Although both the 70 and 30 percent bone scaffolds showed good results, there were important differences between them: “In the broth experiments, the 70 percent scaffold encouraged bone formation much better than the 30 percent scaffold,” said Grayson, “but the 30 percent scaffold is stronger. Since there wasn’t a difference between the two scaffolds in healing the mouse skulls, we are investigating further to figure out which blend is best overall.”

Although the bone taken from cow knees proved successful in the study, as well as being cleared by the FDA for clinical use, the JHU researchers hope to perform further research using powder made from human bones, which are more widely used. They also plan to create more naturally shaped scaffolds and test experimental additives which could encourage the formation of new blood vessels within the scaffolds.

The research paper, “Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration,” has been published in ACS Biomaterials. Other contributing authors included Ben Hung, Bilal Naved, Miguel Dias, Christina Holmes, Jennifer Elisseeff, and Amir Dorafshar.